4,5-Bis(ethoxycarbonyl)-[1,3]dioxolan-2-yl as a new orthoester-type protecting group for the 2′-hydroxyl function in the chemical synthesis of RNA

Article abstract of DOI:10.1081/NCN-200061895

We wish to report 4,5-bis(ethoxycarbonyl)-[1,3]dioxolan-2-yl as a new protecting for the 2′-hydroxyl function. Our cyclic orthoester-type group is compatible with the DMTr strategy for oligonucleotide synthesis. This group was introduced to the 2′-hydroxy

Full text of DOI:10.1081/NCN-200061895

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4,5-BIS(ETHOXYCARBONYL)-[1,3]DIOXOLAN-2-YL AS A
NEW ORTHOESTER-TYPE PROTECTING GROUP FOR THE
2′-HYDROXYL FUNCTION IN THE CHEMICAL SYNTHESIS
OF RNA
Boleslaw Karwowski a b , K. Seio a c & M. Sekine a b
a Department of Life Science , Tokyo Institute of Technology , Japan
b JST (Japan Science and Technology Corporation) , Japan
c Frontier Collaborative Research Institute, Tokyo Institute of Technology , Japan
Published online: 15 Nov 2011.
To cite this article: Boleslaw Karwowski , K. Seio & M. Sekine (2005) 4,5-BIS(ETHOXYCARBONYL)-[1,3]DIOXOLAN-2-YL AS
A NEW ORTHOESTER-TYPE PROTECTING GROUP FOR THE 2′-HYDROXYL FUNCTION IN THE CHEMICAL SYNTHESIS OF RNA,
Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 1111-1114, DOI: 10.1081/NCN-200061895
To link to this article: http://dx.doi.org/10.1081/NCN-200061895
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):1111–1114, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200061895
4,5-BIS(ETHOXYCARBONYL)-[1,3]DIOXOLAN-2-YL AS A NEW
ORTHOESTER-TYPE PROTECTING GROUP FOR THE 2’-HYDROXYL
FUNCTION IN THE CHEMICAL SYNTHESIS OF RNA
5
Boleslaw Karwowski
Department of Life Science, Tokyo Institute of Technology, Japan
and JST (Japan Science and Technology Corporation), Japan
5
K. Seio
Department of Life Science, Tokyo Institute of Technology, Japan and Frontier
Collaborative Research Institute, Tokyo Institute of Technology, Japan
5
M. Sekine
Department of Life Science, Tokyo Institute of Technology, Japan and JST
(Japan Science and Technology Corporation), Japan
5
We wish to report 4,5-bis(ethoxycarbonyl)-[1,3]dioxolan-2-yl as a new protecting for the 2’-
hydroxyl function. Our cyclic orthoester-type group is compatible with the DMTr strategy for
oligonucleotide synthesis. This group was introduced to the 2’-hydroxyl group of appropriately protected
nucleoside derivatives in good yields under mild acidic conditions. Post-synthetic conversion of the moiety
of this protecting group with an amine resulted in formation of a new amide moiety that is more stable
to acid deprotection in aqueous solution, but it can still be easily removed by treatment with acids in
organic solvents. In this article, we also describe the stability of not only the original and modified
protecting groups but also internucleotidic phosphate linkages of protected RNA intermediates under
deprotection conditions.
Keywords RNA Protecting Group, 2-Ethoxy-[1,3]Dioxolane-4,5-Dicarboxylic Acid Diethyl
Ester, Acid Stability
INTRODUCTION
During the last decade, many chemical strategies have been developed for
oligoribonucleotide synthesis.[1] The protecting group of the 2’-hydroxyl function is
one of the most important subjects in RNA synthesis. Although a number of 2’-
hydroxyl protecting groups have been explored, only a few have survived in the
field of nucleoside and nucleotide chemistry, since very limited conditions have
Address correspondence to Dr. Boleslaw Karwowski, CEA Grenoble, LAN/SCIB/DRFMC, 17 Avenue,
Des Martyrs, Grenoble, F-38054, France; Fax: 33-4-38-78-50-90; E-mail: karwowski@cea.fr
1111
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1112
B. Karwowski, K. Seio, and M. Sekine
been required for chemical transformation. For the projected reaction, a protecting
group should fulfill the following requirements:
1. The reagent can be easily prepared.
2. No new chiral centers should be generated.
3. The protecting group should be stable during the projected reaction.
4. Removal of the protecting group must be highly selective without damage of the
other functional groups.[2]
The methoxymethylidene group is well known as the protecting group of
ribonucleoside cis-2’,3’-diol derivatives.[3] From the other point of view, the methanol
is protected by orthoester-type backbone structures. This idea led us to investigate a
new method for the protection of nucleoside 2’-hydroxyl groups with such orthoester
structures. In the literature there are only two examples of the orthoester-type as
protecting groups for RNA synthesis,[4,5] but they have several drawbacks such as
low yields of starting materials and the use of a highly cost co-reagent.[5]
RESULTS AND DISCUSSION
Our attention was paid to cyclic orthoesters which are commercially available.
Compounds 1 and 2 (Figure 1), could be used as introducing reagents for
orthoester functions, as shown in so that they did not generate new chiral centres.
We checked the stability of 3 and 4 under weakly acidic conditions (1-H-
tetrazole), which are necessary for activation of nucleoside phosphoramidites
during the oligonucleotide synthesis by the phosphoramidite strategy. However,
both 3 and 4 were found to be unstable. Because compounds 3 and 4 were not
suitable for the solid-phase synthesis of RNA, our attention was focused on
orthoester derivatives of the D- or L-isomer of tartaric acid. This compound is
suitable for the synthesis of chiral olefins and L-dioxolane nucleosides,[6] the
promising stability of this compound under different conditions led us to study a
new methodology for the protecting group. The starting material 2-ethoxy-
[1,3]dioxolane-4,5-dicarboxylic acid diethyl ester (D- or L-form) was obtained in
excellent yield from the tartaric acid L or D isomer and triethoxyethane.
Since the backbone structures of compounds 6 and 7 were used in our new
strategy for oligonucleotide synthesis, we decided to test their stability under acidic
conditions which are necessary for deprotection of the DMT group from the
FIGURE 1 Synthesis of nucleotides with orthoester function as a potential protecting group.

B. Karwowski, K. Seio, and M. Sekine
1113
FIGURE 2 Synthesis of dinucleotide UpU via 5’-O-DMT-2’-O-[4,5-bis(ethoxycarbonyl)-[1,3]dioxolan-2-yl]uridine
as a substrate. (I) H+, (II) TBAF, (III) DMTrCl, (IV) (Cl)P(N-iPr2)-O(CH2)2CN, (V) 1-H-tetrazol, 2,3-isopropyl-
ideneuridine, (VI) I2/H2O/Pyridine, (VII) NH3aq, (VIII) H+.
5’-hydroxyl function. Compound 6 was stable in a dry solution of DCA and in
acetic acid–water solution.
This data allowed us to make efforts for the synthesis of the nucleoside
derivatives with a [1,3]dioxolane-4,5-dicarboxylic acid diethyl ester skeleton as a
protecting group. Because it is possible to selectively remove the DMT group from
the 5’-O position by DCA in CH2Cl2 we decided to synthesize the phosphoramidite
derivative of 5’-O-DMT-2’-O-[4,5-Bis(ethoxycarbonyl)-[1,3]dioxolan-2-yl]uridine.
This compound was easily obtained by the reaction of an appropriately protected
uridine derivative with the phosphoramidite unit in 60% yield with the isomeric ratio
of 65:35 (Figure 2). We also checked the utility of compound 8 for creation of the
internucleotidic phosphorus bond by synthesis of the dinucleotide UpU.
During the creation of the internucleotide bond, we did not find any
unexpected signals in 31P NMR analysis. The crude product was analyzed by RP
HPLC. This result is promising for future studies because, with the help of this
diamide function, the change of this hydrophilic property enables us to purify them
easily. The difference in the retention time between 10 and the dinucleotide with a
DMT protecting group is big enough to purify each of them. It is an opposite
situation to the current RNA synthesis with the t-butyldimethylsilyl 2’-protecting
group where we did not observe a significant difference in the retention time.
CONCLUSION AND OUTLOOK
The new protecting group of the orthoester-type described in this poster could
be widely used in nucleic acid chemistry as well as carbohydrate chemistry. We
found that the 4,5-bis(ethoxycarbonyl)-[1,3]dioxolan-2-yl 2’-OH protecting group is
stable under acidic conditions with moderate pH values. Fortunately, during our
study, we never observed the loss of this group. It is promising for future research
that our strategy is compatible with the DMT group which is most widely used in
the oligonucleotide synthesis. The stability of this group changed during the
synthesis from ester type 9 to amide-type 10. Additionally, the purification of crude
postsynthetic oligonucleotides should be fast and easy. Future work will continue to
implement this strategy for the solid-phase synthesis of RNA.

1114
REFERENCES
B. Karwowski, K. Seio, and M. Sekine
1. Reese, C.B. The chemical synthesis of oligo- and poly-nucleotides: a personal commentary. Tetrahedron
2002, 58, 8893.
2. Sekine, M.; Hata, T. Cyclic orthoester functions as new protecting groups in nucleosides. J. Am. Chem. Soc.
1983, 105, 2044–20049.
3. Green, L.P.D.; Ravindranathan, R.; Reese, C.B.; Saffihill, R. The synthesis of oligoribonucleotides—VIII: the
preparation of ribonucleoside 2’,5’-bisketals. Tetrahedron 1970, 26, 1031–1041.
4. Sandstrom, S.; Kwiatkowski, A.; Chattopadyaya, J. Chemical synthesis of a pentaribonucleoside tetraphosphate
constituting the 3’-acceptor stem sequence of E. coli tRNAIle using 2’-O-(3-methoxy-1,5-dicarbomethoxypentan-
3-yl)-ribonucleoside building blocks. Application of a new achiral and acid-labile 2’-hydroxyl protecting group
in tRNA synthesis. J. Acta Chem. Scand., B 1985, 39, 273–280.
5. Scaringe, S.A. RNA oligonucleotide synthesis via 5’-silyl-2’-orthoester chemistry. Methods 2001, 23, 206–217.
6. Branalt, J.; Kvarnstrom, I.; Classon, B.; Samuelsson, B. Synthesis of [4,5-bis(hydroxymethyl)-1,3-dioxolan-2-
yl]nucleosides as potential inhibitors of HIV. J. Org. Chem. 1996, 61, 3599–3603.